The present invention relates to the manufacture of carotenoids and biological materials useful therefor.
Astaxanthin is known to be distributed in a wide variety of organisms, such as, for example, animals (e.g., birds, such as, for example, flamingos and scarlet ibis, and fish, such as, for example, rainbow trout and salmon), algae, and microorganisms. It is also recognized that astaxanthin possesses a strong antioxidation property against reactive oxygen species, which suggests a pharmaceutical applicability in protecting living cells against some diseases, such as, for example, cancer. Moreover, from a viewpoint of industrial application, demand for astaxanthin as a coloring reagent is increasing, especially in the industry of farmed fish, such as, for example, salmon, because astaxanthin imparts a distinctive orange-red coloration to the animals that contributes to consumer appeal in the marketplace.
Phaffia rhodozyma is known as a carotenogenic yeast strain that produces astaxanthin. In contrast to other carotenogenic yeast, such as, for example, Rhodotorula, Phaffia rhodozyma (P. rhodozyma) can ferment some sugars such as, for example, D-glucose. This is an important feature from the viewpoint of industrial application. In a recent taxonomic study, the sexual cycle of P. rhodozyma was revealed and its telemorphic state was designated under the name of Xanthophyllomyces dendrorhous (W. I. Golubev; Yeast 11, 101-110, 1995).
In order to obtain hyper-producers of astaxanthin from P. rhodozyma, some strain improvement studies have been conducted. However, in this decade, such efforts have been restricted to employing conventional mutagenesis and protoplast fusion techniques. Recently, Wery et al. developed a host vector system using P. rhodozyma in which multicopies of a non-replicable plasmid were integrated into the genome of P. rhodozyma at the ribosomal DNA locus (Wery et al., Gene, 184, 89-97, 1997). Verdoes et al., International Patent Publication No. WO97/23633, reported the use of improved vectors to transform P. rhodozyma with copies of the three carotenogenic genes that encode enzymes that catalyze the reaction from geranylgeranyl pyrophosphate to beta-carotene.
Many researchers have speculated that astaxanthin might function as an antioxidant in Phaffia rhodozyma because its production is stimulated during the respiration phase of growth rather than during the fermentation phase. In general, reactive oxygen species tend to be generated during the respiration phase as a result of electron overflow in the respiratory chain. Electron overflow in the respiratory chain is caused by an imbalance of electron transfer during reduction of the ubiquinone pool and electron transfer occurring downstream in the respiratory chain. It is speculated that astaxanthin might quench such reactive oxygen species in a manner analogous to superoxide dismutase.
Schroeder and Johnson reported that the respiratory chain of Phaffia rhodozyma shifted from KCN-sensitive respiration to KCN-resistant respiration during the late phase of growth when astaxanthin production was stimulated (J. Biol. Chem., 270, 18374-18379, 1995). The KCN-sensitive respiratory chain, in which an electron from the ubiquinone pool is transferred to complex IV via complex III, is a common electron transfer chain that is found in a wide variety of organisms. It is known that this respiratory chain is inhibited by KCN or antimycin A.
The KCN-resistant respiratory chain is found in both plants and fungi. In this respiratory chain, a mitochondrial membrane protein, alternative oxidase (AOX), plays a substantial role in transferring an electron from the ubiquinone pool to an H2O molecule by using an oxygen molecule as an acceptor. AOX activity is known to be inhibited by n-propyl gallate (n-PG) or salicylhydroxamic acid (SHAM).
In their characterization study of antimycin-sensitive hyper-producers of astaxanthin derived from Phaffia rhodozyma, An et al. speculated that such mutants produced increased amounts of astaxanthin to quench reactive oxygen species, which might be produced by electron overflow from the electron transfer chain (Appl. Env. Microbiol, 55, 116-124, 1989).
This invention was conceived based on the presumption that the biosynthesis of astaxanthin might be upregulated under conditions in which the electron transfer chain is in the reduced state. The reduced state might be induced by addition of a specific inhibitor such as antimycin A, KCN, n-PG or SHAM. The reduced state might also be induced by a mutation that would result in an imbalance in electron transfer.
In accordance with this invention, mutants were obtained that displayed resistance to SHAM. Surprisingly, these mutants displayed 50% higher productivity of astaxanthin than their parent strain.
In the present invention, the cloning of a gene that codes for an alternative oxidase from Phaffia rhodozyma is disclosed. In the present invention, the enzymatic characterization of the expression of the gene in suitable host organisms such as E. coli or Saccharomyces cerevisiae is also disclosed. The cloned gene may be used for the reduction of AOX activity using methods such as, for example, site-directed mutagenesis of promoter sequences or anti-sense methods in a suitable host, such as P. rhodozyma. The effects of gene expression on carotenogenesis can be studied by cultivating transformants in an appropriate medium under appropriate cultivation conditions.
An object of the present invention is a process for producing a carotenoid involving:
(a) culturing a mutant microorganism in culture medium containing an alternative oxidase (AOX) inhibitor, wherein the mutant microorganism produces at least 10% more of the carotenoid compared to the parental stain of the mutant microorganism; and
(b) recovering the carotenoid produced by the mutant microorganism from the culture media.
Another object of the present invention is a process for producing a carotenoid involving:
(a) culturing a microorganism containing a polynucleotide sequence encoding an alternative oxidase (AOX), which polynucleotide sequence has been altered to form a mutant microorganism compared to a parental microorganism containing an unaltered polynucleotide sequence encoding AOX, which mutant microorganism has a reduced level of AOX expression and produces at least 10% more carotenoid as compared to the unaltered parental microorganism; and
(b) recovering the carotenoid produced by the mutant microorganism.
A further object of the present invention is a process for engineering a carotenoid-producing microorganism involving:
(a) selecting a parental microorganism that produces a carotenoid;
(b) culturing the parental microorganism in a culture medium containing an alternative oxidase (AOX) inhibitor; and
(c) selecting a mutant microorganism that grows in the culture medium containing the AOX inhibitor and which microorganism produces at least 10% more of the carotenoid compared to the parental microorganism.
A further object of the present invention is a process for engineering an enhanced carotenoid-producing microorganism involving:
(a) selecting a parental microorganism that produces a carotenoid;
(b) altering a polynucleotide sequence encoding an alternative oxidase (AOX) in the parental microorganism to form a mutant microorganism, which mutant has a reduced level of AOX expression compared to the parental microorganism; and
(c) selecting a mutant microorganism that produces at least 10% more of the carotenoid compared to the parental microorganism.
Another object of the present invention is a recombinantly-produced mutant microorganism produced from a parental carotenoid-producing microorganism having a gene that encodes alternative oxidase (AOX), wherein the gene expression of the AOX in the parental microorganism is altered to produce the mutant microorganism, whereby the efficiency of expression of the AOX in the mutant is reduced compared to the parental microorganism and the mutant produces at least 10% more of a carotenoid compared to the parental microorganism.
A further object of the present invention is an isolated polynucleotide sequence encoding an alternative oxidase derived from a carotenoid-producing microorganism.
Another object of the present invention is a polypeptide having SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 having AOX activity.
A further object of the present invention is a process for producing astaxanthin involving:
(a) cultivating in a culture medium a microorganism transformed with a vector containing an antisense polynucleotide sequence for a native alternate oxidase (AOX) gene in the microorganism, which microorganism produces astaxanthin at a level that is at least 10% greater than a parental strain of the microorganism that is not transformed with the vector; and
(b) collecting the astaxanthin from the microorganism and/or the culture media.
Another object of the present invention is an astaxanthin-producing microorganism having a vector containing an antisense polynucleotide sequence for a native alternate oxidase (AOX) gene in the microorganism, which microorganism produces astaxanthin at a level that is at least 10% greater than a parental strain of the microorganism that is not transformed with the vector.
A further object of the present invention is a process for producing carotenoids, which involves cultivating a microorganism obtained by treating a parent microorganism that produces carotenoids under conditions that induce a reduction in the activity of an alternative oxidase, and selecting a microorganism with enhanced carotenoid productivity. The microorganism utilized in the process of the present invention may be a mutant strain in which carotenoid productivity is enhanced due to an altered resistance against the alternative oxidase inhibitor. The process according to the present invention can be practiced by using a microorganism belonging to the kingdom of Protista or Fungi, preferably to the genus Synechococcus, Synechocystis, Haematococcus, Dunaliella, Phaffia, Xanthophyllomyces, Neurospora, Rhodotorula, Blakeslea, or Phycomyces, more preferably the microorganism may be Phaffia rhodozyma or Xanthophyllomyces dendrorhous. The alternative oxidase inhibitor used in the present invention may be selected from n-propyl gallate and salicylhydroxamic acid.
Another object of the present invention is a method for establishing a mutant strain capable of producing carotenoids at an enhanced level relative to the parent microorganism. The method involves cultivating a microorganism that produces carotenoids under conditions that reduce the activity of an alternative oxidase and selecting for a microorganism capable of producing carotenoids at a higher level than the parent microorganism. The conditions that reduce alternative oxidase activity may involve the presence of an alternative oxidase inhibitor. The alternative oxidase inhibitor may be selected from n-propyl gallate and salicylhydroxamic acid. The microorganism may belong to the kingdom of Protista or Fungi, preferably to the genus Synechococcus, Synechocystis, Haematococcus, Dunaliella, Phaffia, Xanthophyllomyces, Neurospora, Rhodotorula, Blakeslea, or Phycomyces, more preferably the microorganism may be Phaffia rhodozyma or Xanthophyllomyces dendrorhous. 
A further object of the present invention is a mutant strain capable of producing carotenoids at an enhanced level, relative to a parent microorganism, obtained by the method described above. The mutant may be more specifically characterized in that it can grow in a medium containing 0.3 to 0.45 mg/ml of SHAM at a growth rate comparable to the growth rate in a medium that does not contain SHAM.
An embodiment of the present invention is a SHAM-resistant mutant derived from Phaffia rhodozyma ATCC 96594. SHAM-resistant mutant strains have been deposited at the DSMZ (Deutsche Sammlung der Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany) under the designations DSM 13429, DSM 13430 and DSM 13431, on Apr. 3, 2000.
The microorganism used in the process of the present invention may be a recombinant microorganism in which gene expression of the alternative oxidase has been altered to reduce overall efficiency compared to the parent microorganism. Accordingly, another object of the invention is a recombinant microorganism capable of producing carotenoids at an enhanced level relative to the host microorganism, whose gene expression of the alternative oxidase has been altered to reduce overall efficiency compared to the host microorganism. The alternative oxidase gene expression of the host microorganism may be altered by using a genetic technique, such as, for example, antisense technology, site-directed mutagenesis, chemical mutagenesis, and other commonly used mutagenic techniques. The microorganism used for this purpose may belong to the kingdom of Protista or Fungi, preferably to the genus Synechococcus, Synechocystis, Haematococcus, Dunaliella, Phaffia, Xanthophyllomyces, Neurospora, Rhodotorula, Blakeslea, or Phycomyces, more preferably the microorganism may be Phaffia rhodozyma or Xanthophyllomyces dendrorhous, most preferably the microorganism may be one of the deposited strains, DSM 13429, DSM 13430 and DSM 13431.
A further object of the invention is a recombinant DNA sequence that encodes an alternative oxidase derived from a microorganism capable of producing carotenoids. The recombinant DNA may preferably be obtained from a microorganism which belongs to the kingdom of Protista or Fungi, more preferably the genus Synechococcus, Synechocystis, Haematococcus, Dunaliella, Phaffia, Xanthophyllomyces, Neurospora, Rhodotorula, Blakeslea, or Phycomyces, even more preferably the microorganism may be Phaffia rhodozyma or Xanthophyllomyces dendrorhous, most preferably the microorganism may be one of the deposited strains, DSM 13429, DSM 13430 and DSM 13431. The recombinant DNA sequence may be that identified by SEQ ID NO: 2 or may be a sequence having identity with SEQ ID NO: 2 higher than 55%, more preferably higher than 75%, such as, for example, higher than 95%.
The recombinant DNA sequence may be more specifically characterized in that it encodes (a) the enzyme having the amino acid sequence shown in SEQ ID NO: 1, or (b) a variant of the enzyme having the amino acid sequence shown in SEQ ID NO: 1 selected from (i) an allelic variant, and (ii) an enzyme having one or more (such as 1-50, 2-40, 3-30, 4-20 or 5-10) amino acid additions, insertions, deletions and/or substitutions and still having the stated enzymatic activity. The isolated DNA sequence may be derived from a gene of Phaffia rhodozyma and selected from (i) the DNA sequence shown in SEQ ID NO: 2, (ii) an isocoding or allelic variant of the DNA sequence shown in SEQ ID NO: 2, and (iii) a derivative of a DNA sequence shown in SEQ ID NO: 2, with additions, insertions, deletions and/or substitutions of one or more nucleotide(s), and coding for a polypeptide having the stated enzymatic activity.
Another object of the invention is the use of the recombinant DNA to transform a host microorganism. A convenient form of the recombinant DNA may be a vector. The recombinant microorganism obtained by use of the recombinant DNA should be capable of decreasing the enzyme activity of the alternative oxidase. The host microorganism transformed with the recombinant DNA may be useful in improving carotenoid production, in particular astaxanthin. Accordingly, another object of the invention is such a recombinant microorganism.
A further object of the invention is a method for the biological production of carotenoids, which involves introducing the recombinant DNA, as set forth above, into an appropriate host microorganism and cultivating the resulting recombinant microorganism under conditions conducive to the production of carotenoids. This method may preferably be applied to the biological production of astaxanthin.